Abstract Staphylococcusaureusisthemostcommoninvasivehumanpathogenwithassociatedinfections originatinginmultiplesettingsandpatienttypes.Infectionsduetomethicillin-resistantS.aureus (MRSA) increase patient morbidity and mortality in part due to limited therapeutic options and increasingantibioticresistancetoprimaryantibiotics.Recentevidencesuggeststhatcertainb?- lactams, traditionally considered inactive against MRSA, can enhance clinical efficacy against bothMRSAandMSSAinfectionsbysynergizingwithdaptomycin(DAP)andcationichostdefense peptides(HDPs)ofwhitecellandplateletorigins.Mechanistically,thisapparent?synergy?was initially attributed to enhancement of binding of these peptide antibiotics to the cell membrane targets in the presence of b?-lactams. Other mechanisms appear to play a role, and we have identifiedthatdiscriminativeinhibitionofpenicillin-bindingproteins(PBPs)withb?-lactamsresults indifferentialdaptomycinsynergy.Ourpreliminaryscreeningindicatesthatblockadeoftheaction ofPBP-1(eitherspecificallyorpromiscuously)isessentialtothisDAP-b?-lactamsynergyoutcome. Giventhesefindings,wepositthatb?-lactamswitheitherselectiveornonselectivePBP-1blocking activity provide multi-mechanistic and synergistic killing against S. aureus when used in combination with DAP. To test this hypothesis, we will conduct studies using three integrated Aims. In Aim 1, we identify mechanistic interactions of PBP inhibition underlying DAP/HDP synergy with b?-lactams through manipulating PBP function and defining the compensatory impactsofDAP-b?-lactamcombinationsonkeycellwallandcellmembranefunctionalmetricsthat traditionallylinktoantimicrobialpotency.InAim2,wedetermineoptimalb?-lactamstrategiesin combination with DAP or HDPs against MRSA using discriminative in vitro modeling with the hollowfiberbioreactorsystem.Aim3establishestheoptimalcombinedb?-lactam-DAPtreatment regimens in vivo, utilizing a prototypical endovascular MRSA infection model, experimental infectiveendocarditis.Attheconclusionofthesestudies,ourresultswillidentifynewmechanisms associated with DAP?s lethal pathway and provide a potentially unique option for rescuing continueduseofDAPinclinicalpractice.Ultimately,thisprojectmaywellprovideclinicianswith improvedtreatmentstrategieswithsmarttargetedcombinationsforcomplexandrefractoryMRSA infections.